high-quality-seizure-like-activity-from-acute-brain-slices-using

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures ...

Brigham Young University

Voltage-gated sodium channels (Nav) are essential for the initiation and propagation of action potentials in neurons. Of the nine human channel subtypes, Nav1.1, Nav1.2 and Nav1.6 are prominently expressed in the adult central nervous system (CNS). All three of these sodium channel subtypes are sensitive to block by the neurotoxin tetrodotoxin (TTX), with TTX being almost equipotent on all three subtypes. In the present study we have used TTX to determine the fractional block of Nav channels required to impair action potential firing in pyramidal neurons and reduce network seizure-like activity. Using automated patch-clamp electrophysiology, we first determined the ICs of TTX on mouse Nav1.1, Nav1.2 and Nav1.6 channels expressed in HEK cells, demonstrating this to be consistent with previously published data on human orthologs. We then compared this data to the potency of block of Nav current measured in pyramidal neurons from neocortical brain slices. Interestingly, we found that it requires nearly 10-fold greater concentration of TTX over the IC to induce significant block of action potentials using a current-step protocol. In contrast, concentrations near the IC resulted in a significant reduction in AP firing and increase in rheobase using a ramp protocol. Surprisingly, a 20% reduction in action potential generation observed with 3 nM TTX resulted in significant block of seizure-like activity in the 0 Mg model of epilepsy. Additionally, we found that approximately 50% block in pyramidal cell intrinsic excitability is sufficient to completely block all seizure-like events. Furthermore, we also show that the anticonvulsant drug phenytoin blocked seizure-like events in a manner similar to TTX. These data serve as a critical starting point in understanding how fractional block of Nav channels affect intrinsic neuronal excitability and seizure-like activity. It further suggests that seizures can be controlled without significantly compromising intrinsic neuronal activity and determines the required fold over IC for novel and clinically relevant Nav channel blockers to produce efficacy and limit side effects.

Pharmacological determination of the fractional block of Nav channels required to impair neuronal excitability and  seizures.

The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, halorhodopsin. We recorded extracellular K concentration ([K]) in neocortical brain slices prepared from young adult mice (both sexes) which express halorhodopsin in pyramidal cells. Strong halorhodopsin activation induced a pronounced drop in [K] that persisted for the duration of illumination. Pharmacological blockade of K channels reduced the amplitude of this drop, indicating that it represents K redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl directly, and K secondarily. When the illumination period ended, a rebound surge in extracellular [K] developed over tens of seconds, partly reflecting the previous inward redistribution of K, but additionally driven by clearance of Cl coupled to K by the potassium-chloride cotransporter, KCC2. The drop in [K] during light activation leads to a small (2-3 mV) hyperpolarization also of other cells that do not express halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of halorhodopsin causes cortical spreading depolarizations (CSDs), both  and  This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K] is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally. Halorhodopsin is a light-activated electrogenic chloride pump, which has been widely used to inhibit neurons optogenetically. Here, we demonstrate three previously unrecognized consequences of its use: (1) intense activation leads to secondary movement of K ions into the cells; (2) the resultant drop in extracellular [K] reduces excitability also in other, nonexpressing cells; and (3) intense persistent halorhodopsin activation can trigger cortical spreading depolarization (CSD). Halorhodopsin-induced CSDs can occur when neurons are hyperpolarized and extracellular [K] is low. This contrasts with the most widely used experimental models that trigger CSDs with high [K]. Both models, however, are consistent with the hypothesis that CSDs arise following net inward ionic movement into the principal neuron population.

Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization.

Investigations into seizure initiation, in recent years, have focused almost entirely upon alterations of interneuronal function, chloride homeostasis, and extracellular potassium levels. In contrast, little attention has been directed toward a possible role of dendritic plateau potentials in the actual ictogenic transition, despite a substantial literature dating back 40 years regarding its importance generally in epilepsy. Here, we argue that an increase in dendritic excitability, coordinated across the population of pyramidal cells, is a key stage in ictogenesis.

Synergistic Positive Feedback Mechanisms Underlying Seizure Initiation.

We recently reported that strong activation of the optogenetic chloride pump, halorhodopsin leads to a secondary redistribution of K ions into the cell, through tonically open, "leak" K channels. Here we show that this effect is not unique to halorhodopsin but is also seen with activation of another electrogenic ion pump, archaerhodopsin. The two opsins differ however in the size of the rebound rise in extracellular potassium, [K ] , after the end of activation, which is far larger with halorhodopsin than for archaerhodopsin activation. Multiple linear regression modeling indicates that the variance in the postillumination surge in [K ] was explained both by the size of the preceding, illumination-induced drop in [K ] and also by the type of opsin. These data provide additional support for the hypothesis that intense chloride-loading of cells, as occurs naturally following intense bursts of GABAergic synaptic bombardment, or artificially following halorhodopsin activation, is followed by extrusion of both Cl and K coupled together. We discuss this with respect to the pattern of [K ] rise that occurs at the onset of seizure-like events.

Optogenetic ion pumps differ with respect to the secondary pattern of K redistribution.

Seizures are generally associated with epilepsy but may also be a symptom of many other neurological conditions. A hallmark of a seizure is the intensity of the local neuronal activation, which can drive large-scale gene transcription changes. Such changes in the transcriptional profile likely alter neuronal function, thereby contributing to the pathological process. Therefore, there is a strong clinical imperative to characterize how gene expression is changed by seizure activity. To this end, we developed a simplified ex vivo technique for studying seizure-induced transcriptional changes. We compared the RNA sequencing profile in mouse neocortical tissue with up to 3 h of epileptiform activity induced by 4-aminopyridine (4AP) relative to control brain slices not exposed to the drug. We identified over 100 genes with significantly altered expression after 4AP treatment, including multiple genes involved in MAPK, TNF, and neuroinflammatory signaling pathways, all of which have been linked to epilepsy previously. Notably, the patterns in male and female brain slices were almost identical. Various immediate early genes were among those showing the largest upregulation. The set of down-regulated genes included ones that might be expected either to increase or to decrease neuronal excitability. In summary, we found the seizure-induced transcriptional profile complex, but the changes aligned well with an analysis of published epilepsy-associated genes. We discuss how simple models may provide new angles for investigating seizure-induced transcriptional changes.

RNA Sequencing Demonstrates Ex Vivo Neocortical Transcriptomic Changes Induced by Epileptiform Activity in Male and Female Mice.

Temporal lobe epilepsy (TLE) is a type of focal epilepsy characterized by spontaneous recurrent seizures originating from the hippocampus. The epigenetic reprogramming hypothesis of epileptogenesis suggests that the development of TLE is associated with alterations in gene transcription changes resulting in a hyperexcitable network in TLE. DNA 5-methylcytosine (5-mC) is an epigenetic mechanism that has been associated with chronic epilepsy. However, the contribution of 5-hydroxymethylcytosine (5-hmC), a product of 5-mC demethylation by the Ten-Eleven Translocation (TET) family proteins in chronic TLE is poorly understood. 5-hmC is abundant in the brain and acts as a stable epigenetic mark altering gene expression through several mechanisms. Here, we found that the levels of bulk DNA 5-hmC but not 5-mC were significantly reduced in the hippocampus of human TLE patients and in the kainic acid (KA) TLE rat model. Using 5-hmC hMeDIP-sequencing, we characterized 5-hmC distribution across the genome and found bidirectional regulation of 5-hmC at intergenic regions within gene bodies. We found that hypohydroxymethylated 5-hmC intergenic regions were associated with several epilepsy-related genes, including Gal, SV2, and Kcnj11 and hyperdroxymethylation 5-hmC intergenic regions were associated with Gad65, TLR4, and Bdnf gene expression. Mechanistically, Tet1 knockdown in the hippocampus was sufficient to decrease 5-hmC levels and increase seizure susceptibility following KA administration. In contrast, Tet1 overexpression in the hippocampus resulted in increased 5-hmC levels associated with improved seizure resiliency in response to KA. These findings suggest an important role for 5-hmC as an epigenetic regulator of epilepsy that can be manipulated to influence seizure outcomes.

Alterations in DNA 5-hydroxymethylation patterns in the hippocampus of an experimental model of chronic epilepsy.

Much of what has been discovered concerning neurophysiological mechanisms can be credited to ex vivo biomedical experiments. Beyond these discoveries, ex vivo research techniques have enhanced the global understanding of human physiology and pathology in almost every biomedical specialty. Naturally, ex vivo experiments are among the most desired methods of research, particularly in the field of neuroscience. Ex vivo experiment platforms may be purchased commercially. However, their substantial cost and sometimes limited availability can render them inaccessible to many research labs. Moreover, these manufactured systems are often rigid in function with no possibility of customization, severely narrowing their capabilities. However, developing essential components for ex vivo laboratory systems with a fused deposition modeling printer provides a practical solution to each of these obstacles. Here, we provide the designs and construction process for an easily accessible, highly adaptable recording stage with modifiable submersion chambers using a 3D printer for a total cost under $15.00. With the versatility afforded by the exchangeable custom chambers, the system may be used to conduct research on a variety of ex vivo tissue preparations, paving the way for novel research.

Article	Year
Pharmacological determination of the fractional block of Nav channels required to impair neuronal excitability and seizures. Frontiers in cellular neuroscience\| PubMed ID: 36246527	2022
Indirect Effects of Halorhodopsin Activation: Potassium Redistribution, Nonspecific Inhibition, and Spreading Depolarization. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 36639898	2023
Synergistic Positive Feedback Mechanisms Underlying Seizure Initiation. Epilepsy currents\| PubMed ID: 36923333	2023
Optogenetic ion pumps differ with respect to the secondary pattern of K redistribution. Physiological reports\| PubMed ID: 37537145	2023
RNA Sequencing Demonstrates Ex Vivo Neocortical Transcriptomic Changes Induced by Epileptiform Activity in Male and Female Mice. eNeuro\| PubMed ID: 38664009	2024
Alterations in DNA 5-hydroxymethylation patterns in the hippocampus of an experimental model of chronic epilepsy. Neurobiology of disease\| PubMed ID: 39142613	2024
An Open-Source 3D-Printed Recording Stage with Customizable Chambers for Ex Vivo Experiments. eNeuro\| PubMed ID: 39197950	2024

R. Ryley Parrish

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